Protein formulation

ABSTRACT

A stable lyophilized protein formulation is described which can be reconstituted with a suitable diluent to generate a high protein concentration reconstituted formulation which is suitable for subcutaneous administration. For example, anti-IgE and anti-HER2 antibody formulations have been prepared by lyophilizing these antibodies in the presence of a lyoprotectant. The lyophilized mixture thus formed is reconstituted to a high protein concentration without apparent loss of stability of the protein.

This is a continuation of U.S. patent application Ser. No. 13/156,113filed Jun. 8, 2011, which is a continuation of U.S. patent applicationSer. No. 12/698,347 filed Feb. 2, 2010, which is a continuation of U.S.patent application Ser. No. 11/441,994 filed May 26, 2006, (now U.S.Pat. No. 7,682,609), which is a continuation of U.S. patent applicationSer. No. 09/273,230 filed Mar. 18, 1999, (now abandoned) which is adivisional application of U.S. patent application Ser. No. 08/615,369filed Mar. 14, 1996 (now U.S. Pat. No. 6,267,958 B1), which claimspriority under 35 U.S.C. Section 119(e) and the benefit of U.S.Provisional Application Ser. No. 60/029,182, filed Jul. 27, 1995, theentire disclosures of which are incorporated herein by reference intheir entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is directed to a lyophilized protein formulation. Inparticular, it relates to a stable lyophilized protein formulation whichcan be reconstituted with a diluent to generate a stable reconstitutedformulation suitable for subcutaneous administration.

2. Description of Related Disclosures

In the past ten years, advances in biotechnology have made it possibleto produce a variety of proteins for pharmaceutical applications usingrecombinant DNA techniques. Because proteins are larger and more complexthan traditional organic and inorganic drugs (i.e. possessing multiplefunctional groups in addition to complex three-dimensional structures),the formulation of such proteins poses special problems. For a proteinto remain biologically active, a formulation must preserve intact theconformational integrity of at least a core sequence of the protein'samino acids while at the same time protecting the protein's multiplefunctional groups from degradation. Degradation pathways for proteinscan involve chemical instability (i.e. any process which involvesmodification of the protein by bond formation or cleavage resulting in anew chemical entity) or physical instability (i.e. changes in the higherorder structure of the protein). Chemical instability can result fromdeamidation, racemization, hydrolysis, oxidation, beta elimination ordisulfide exchange. Physical instability can result from denaturation,aggregation, precipitation or adsorption, for example. The three mostcommon protein degradation pathways are protein aggregation, deamidationand oxidation. Cleland et al. Critical Reviews in Therapeutic DrugCarrier Systems 10(4): 307-377 (1993).

Freeze-drying is a commonly employed technique for preserving proteinswhich serves to remove water from the protein preparation of interest.Freeze-drying, or lyophilization, is a process by which the material tobe dried is first frozen and then the ice or frozen solvent is removedby sublimation in a vacuum environment. An excipient may be included inpre-lyophilized formulations to enhance stability during thefreeze-drying process and/or to improve stability of the lyophilizedproduct upon storage. Pikal, M. Biopharm. 3(9)26-30 (1990) and Arakawaet al. Pharm. Res. 8(3):285-291 (1991). It is an object of the presentinvention to provide a lyophilized protein formulation which is stableupon storage and delivery. It is a further object to provide a stablereconstituted protein formulation which is suitable for subcutaneousadministration. In certain embodiments, it is an object to provide amulti-use formulation which is stable for at least the time over whichit will be administered to a patient.

SUMMARY OF THE INVENTION

This invention is based on the discovery that a stable lyophilizedprotein formulation can be prepared using a lyoprotectant (preferably asugar such as sucrose or trehalose), which lyophilized formulation canbe reconstituted to generate a stable reconstituted formulation having aprotein concentration which is significantly higher (e.g. from about2-40 times higher, preferably 3-10 times higher and most preferably 3-6times higher) than the protein concentration in the pre-lyophilizedformulation. In particular, while the protein concentration in thepre-lyophilized formulation may be 5 mg/mL or less, the proteinconcentration in the reconstituted formulation is generally 50 mg/mL ormore. Such high protein concentrations in the reconstituted formulationare considered to be particularly useful where the formulation isintended for subcutaneous administration. Despite the very high proteinconcentration in the reconstituted formulation, it has been found thatthe reconstituted formulation is stable (i.e. fails to displaysignificant or unacceptable levels of chemical or physical instabilityof the protein) at 2-8° C. for at least about 30 days. In certainembodiments, the reconstituted formulation is isotonic. In spite of theuse of lower concentrations of the lyoprotectant to achieve suchisotonic formulations upon reconstitution, it was discovered herein thatthe protein in the lyophilized formulation essentially retains itsphysical and chemical stability and integrity upon lyophilization andstorage.

When reconstituted with a diluent comprising a preservative (such asbacteriostatic water for injection, BWFI), the reconstituted formulationmay be used as a multi-use formulation. Such a formulation is useful,for example, where the patient requires frequent subcutaneousadministrations of the protein to treat a chronic medical condition. Theadvantage of a multi-use formulation is that it facilitates ease of usefor the patient, reduces waste by allowing complete use of vialcontents, and results in a significant cost savings for the manufacturersince several doses are packaged in a single vial (lower filling andshipping costs).

Based on the observations described herein, in one aspect the inventionprovides a stable isotonic reconstituted formulation comprising aprotein in an amount of at least about 50 mg/mL and a diluent, whichreconstituted formulation has been prepared from a lyophilized mixtureof a protein and a lyoprotectant, wherein the protein concentration inthe reconstituted formulation is about 2-40 times greater than theprotein concentration in the mixture before lyophilization.

In another embodiment, the invention provides a stable reconstitutedformulation comprising an antibody in an amount of at least about 50mg/mL and a diluent, which reconstituted formulation has been preparedfrom a lyophilized mixture of an antibody and a lyoprotectant, whereinthe antibody concentration in the reconstituted formulation is about2-40 times greater than the antibody concentration in the mixture beforelyophilization.

The ratio of lyoprotectant:protein in the lyophilized formulation of thepreceding paragraphs depends, for example, on both the protein andlyoprotectant of choice, as well as the desired protein concentrationand isotonicity of the reconstituted formulation. In the case of a fulllength antibody (as the protein) and trehalose or sucrose (as thelyoprotectant) for generating a high protein concentration isotonicreconstituted formulation, the ratio may, for example, be about 100-1500mole trehalose or sucrose:1 mole antibody.

Generally, the pre-lyophilized formulation of the protein andlyoprotectant will further include a buffer which provides theformulation at a suitable pH, depending on the protein in theformulation. For this purpose, it has been found to be desirable to usea histidine buffer in that, as demonstrated below, this appears to havelyoprotective properties.

The formulation may further include a surfactant (e.g. a polysorbate) inthat it has been observed herein that this can reduce aggregation of thereconstituted protein and/or reduce the formation of particulates in thereconstituted formulation. The surfactant can be added to thepre-lyophilized formulation, the lyophilized formulation and/or thereconstituted formulation (but preferably the pre-lyophilizedformulation) as desired.

The invention further provides a method for preparing a stable isotonicreconstituted formulation comprising reconstituting a lyophilizedmixture of a protein and a lyoprotectant in a diluent such that theprotein concentration in the reconstituted formulation is at least 50mg/mL, wherein the protein concentration in the reconstitutedformulation is about 2-40 times greater than the protein concentrationin the mixture before lyophilization.

In yet a further embodiment, the invention provides a method forpreparing a formulation comprising the steps of: (a) lyophilizing amixture of a protein and a lyoprotectant; and (b) reconstituting thelyophilized mixture of step (a) in a diluent such that the reconstitutedformulation is isotonic and stable and has a protein concentration of atleast about 50 mg/mL. For example, the protein concentration in thereconstituted formulation may be from about 80 mg/mL to about 300 mg/mL.Generally, the protein concentration in the reconstituted formulation isabout 2-40 times greater than the protein concentration in the mixturebefore lyophilization.

An article of manufacture is also provided herein which comprises: (a) acontainer which holds a lyophilized mixture of a protein and alyoprotectant; and (b) instructions for reconstituting the lyophilizedmixture with a diluent to a protein concentration in the reconstitutedformulation of at least about 50 mg/mL. The article of manufacture mayfurther comprise a second container which holds a diluent (e.g.bacteriostatic water for injection (BWFI) comprising an aromaticalcohol).

The invention further provides a method for treating a mammal comprisingadministering a therapeutically effective amount of a reconstitutedformulation disclosed herein to a mammal, wherein the mammal has adisorder requiring treatment with the protein in the formulation. Forexample, the formulation may be administered subcutaneously.

One useful anti-HER2 antibody pre-lyophilized formulation as discoveredin the experiments detailed below was found to comprise anti-HER2 inamount from about 5-40 mg/mL (e.g. 20-30 mg/mL) and sucrose or trehalosein an amount from about 10-100 mM (e.g. 40-80 mM), a buffer (e.g.histidine, pH 6 or succinate, pH 5) and a surfactant (e.g. apolysorbate). The lyophilized formulation was found to be stable at 40°C. for at least 3 months and stable at 30° C. for at least 6 months.This anti-HER2 formulation can be reconstituted with a diluent togenerate a formulation suitable for intravenous administrationcomprising anti-HER2 in an amount from about 10-30 mg/mL which is stableat 2-8° C. for at least about 30 days. Where higher concentrations ofthe anti-HER2 antibody are desired (for example where subcutaneousdelivery of the antibody is the intended mode of administration to thepatient), the lyophilized formulation may be reconstituted to yield astable reconstituted formulation having a protein concentration of 50mg/mL or more.

One desirable anti-IgE antibody pre-lyophilized formulation discoveredherein has anti-IgE in amount from about 5-40 mg/mL (e.g. 20-30 mg/mL)and sucrose or trehalose in an amount from about 60-300 mM (e.g. 80-170mM), a buffer (preferably histidine, pH 6) and a surfactant (such as apolysorbate). The lyophilized anti-IgE formulation is stable at 30° C.for at least 1 year. This formulation can be reconstituted to yield aformulation comprising anti-IgE in an amount from about 15-45 mg/mL(e.g. 15-25 mg/mL) suitable for intravenous administration which isstable at 2-8° C. for at least 1 year. Alternatively, where higherconcentrations of anti-IgE in the formulation are desired, thelyophilized formulation can be reconstituted in order to generate astable formulation having an anti-IgE concentration of ≧50 mg/mL.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of reconstitution volume on the stability oflyophilized rhuMAb HER2. The lyophilized formulation was prepared from apre-lyophilization formulation comprising 25 mg/mL protein, 60 mMtrehalose, 5 mM sodium succinate, pH 5.0, and 0.01% Tween 20™. Thelyophilized cake was incubated at 40° C. and then reconstituted with 4.0(∘) or 20.0 mL (●) of BWFI. The fraction of intact protein in thereconstituted formulation was measured by native size exclusionchromatography and defined as the peak area of the native proteinrelative to the total peak area including aggregates.

FIG. 2 illustrates the effect of trehalose concentration on thestability of lyophilized rhuMAb HER2. The protein was lyophilized at 25mg/mL in 5 mM sodium succinate, pH 5.0 (circles) or 5 mM histidine, pH6.0 (squares) and trehalose concentrations ranging from 60 mM (360 molarratio) to 200 mM (1200 molar ratio). The lyophilized protein wasincubated at 40° C. for either 30 days (closed symbols) or 91 days (opensymbols). The amount of intact protein was measured after reconstitutionof the lyophilized protein with 20 mL BWFI.

FIG. 3 demonstrates the effect of trehalose concentration on the longterm stability of lyophilized rhuMAb HER2 stored at 40° C. The proteinwas lyophilized at either 25 mg/mL in 5 mM sodium succinate, pH 5.0,0.01% Tween 20™, and 60 mM trehalose (▪) or 5 mM histidine, pH 6.0,0.01% Tween 20™, and 60 mM trehalose (□) or 21 mg/mL in 10 mM sodiumsuccinate, pH 5.0, 0.2% Tween 20™0 and 250 mM trehalose (●). Thelyophilized protein was incubated at 40° C. and then reconstituted with20 mL of BWFI. The amount of intact protein was measured afterreconstitution.

FIG. 4 shows the stability of rhuMAb HER2 lyophilized in 38.4 mMmannitol (7 mg/mL), 20.4 mM sucrose (7 mg/mL), 5 mM histidine, pH 6.0,0.01% Tween 20™. The lyophilized protein was incubated at 40° C. andthen reconstituted with either 4.0 mL (∘) or 20 mL (●) of BWFI. Theamount of intact protein was measured after reconstitution.

FIG. 5 demonstrates stability of reconstituted rhuMAb HER2 lyophilizedin 5 mM sodium succinate, pH 5.0, 60 mM trehalose, 0.01% Tween 20™.Samples were reconstituted with either 4.0 mL (squares) or 20.0 mL(circles) of BWFI (20 mL:0.9% benzyl alcohol; 4 mL:1.1% benzyl alcohol)and then stored at 5° C. (solid symbols) or 25° C. (open symbols). The %native protein was defined as the peak area of the native (not degraded)protein relative to the total peak area as measured by cation exchangechromatography.

FIG. 6 shows stability of reconstituted rhuMAb HER2 lyophilized in 5 mMhistidine, pH 6.0, 60 mM trehalose, 0.01% Tween 20. Samples werereconstituted with either 4.0 mL (squares) or 20.0 mL (circles) of BWFI(20 mL:0.9% benzyl alcohol; 4 mL:1.1% benzyl alcohol) and then stored at5° C. (solid symbols) or 25° C. (open symbols). The % native protein wasdefined as the peak area of the native (not degraded) protein relativeto the total peak area as measured by cation exchange chromatography.

FIG. 7 reveals stability of reconstituted rhuMAb HER2 lyophilized in 5mM histidine, pH 6.0, 38.4 mM mannitol, 20.4 mM sucrose, 0.01% Tween 20.Samples were reconstituted with either 4.0 mL (squares) or 20.0 mL(circles) of BWFI (20 mL:0.9% benzyl alcohol; 4 mL:1.1% benzyl alcohol)and then stored at 5° C. (solid symbols) or 25° C. (open symbols). The %native protein was defined as the peak area of the native (not degraded)protein relative to the total peak area as measured by cation exchangechromatography.

FIG. 8 shows stability of reconstituted rhuMAb HER2 lyophilized in 10 mMsodium succinate, pH 5.0, 250 mM trehalose, 0.2% Tween 20. Samples werereconstituted with 20.0 mL of BWFI (0.9% benzyl alcohol) and then storedat 5° C. (●) or 25° C. (∘). The % native protein was defined as the peakarea of the native (not degraded) protein relative to the total peakarea as measured by cation exchange chromatography.

FIG. 9 shows aggregation of rhuMAb E25 formulated into buffers rangingfrom pH 5 to pH 7 at 10 mM buffer concentration and 5 mg/mL antibodyconcentration. Samples were lyophilized and assayed at time zero andafter 4 weeks, 8 weeks, and 52 weeks of storage at 2-8° C. The bufferswere: potassium phosphate pH 7.0 (∘); sodium phosphate pH 7.0 (□);histidine pH 7.0 (⋄); sodium succinate pH 6.5 (●); sodium succinate pH6.0 (▪); sodium succinate pH 5.5 (□); and sodium succinate pH 5.0 (▴).

FIG. 10 depicts aggregation of rhuMAb E25 lyophilized in 5 mM histidinebuffer at both pH 6 and pH 7 and assayed following storage as follows.The buffer was at: pH 6.0 stored at 2-8° C. (∘); pH 6 stored at 25° C.(□); pH 6 stored at 40° C. (⋄); pH 7 stored at 2-8° C. (●); pH 7 storedat 25° C. (▪); and pH 7 stored at 40° C. (□).

FIG. 11 illustrates aggregation of 5 mg/mL rhuMAb E25 formulated into 10mM sodium succinate at pH 5.0 with lyoprotectant added at aconcentration of 275 mM (isotonic). The lyoprotectants were: control, nolyoprotectant (∘); mannitol (□); lactose (⋄); maltose (●); trehalose(▪); and sucrose (□). Samples were lyophilized and assayed at time zeroand after 4 weeks, 8 weeks, and 52 weeks of storage at 2-8° C.

FIG. 12 shows aggregation of 5 mg/mL rhuMAb E25 formulated into 10 mMsodium succinate at pH 5.0 with lyoprotectant added at a concentrationof 275 mM (isotonic). The lyoprotectants were: control, no lyoprotectant(∘); mannitol (□); lactose (⋄); maltose (●); trehalose (▪); and sucrose(□). Samples were lyophilized and assayed at time zero and after 4weeks, 8 weeks, and 52 weeks of storage at 40° C.

FIG. 13 depicts hydrophobic interaction chromatography of 20 mg/mLrhuMAb E25 lyophilized in histidine buffer at pH 6 with an isotonicconcentration (i.e. 275 mM) of lactose stored for 24 weeks at 2-8, 25 or40° C. and reconstituted to 20 mg/mL.

FIG. 14 shows hydrophobic interaction chromatography of 20 mg/mL rhuMAbE25 lyophilized in histidine buffer at pH 6 stored for 24 weeks at 2-8,25 or 40° C. and reconstituted to 20 mg/mL.

FIG. 15 illustrates hydrophobic interaction chromatography of 20 mg/mLrhuMAb E25 lyophilized in histidine buffer at pH 6 with an isotonicconcentration (i. e. 275 mM) of sucrose and stored for 24 weeks at 2-8,25 or 40° C. and reconstituted to 20 mg/mL.

FIG. 16 illustrates the effect of sugar concentration on rhuMAb E25formulated at 20 mg/mL in 5 mM histidine at pH 6.0. Sucrose (●) andtrehalose (□) were added to the formulation at molar ratios ranging from0 to 2010 (isotonic) (see Table 1 below). Samples were lyophilized andassayed after 12 weeks of storage at 50° C.

TABLE 1 Moles of Sugar: Sugar conc. E25 antibody (mM) 0 0 260 34.4 38051.6 510 68.8 760 103.1 1020 137.5 1530 206.3 2010 275

FIG. 17 reveals aggregation of rhuMAb E25 formulated at 25 mg/mL into 5mM histidine at pH 6 with 85 mM sucrose (∘); 85 mM trehalose (□); 161 mMsucrose (□) or 161 mM trehalose (▴). Samples were lyophilized and storedat 2-8° C. followed by reconstitution with 0.9% benzyl alcohol to 100mg/mL antibody in 20 mM histidine at pH 6 with isotonic (340 mM) andhypertonic (644 mM) sugar concentration.

FIG. 18 shows aggregation of rhuMAb E25 formulated at 25 mg/mL into 5 mMhistidine at pH 6 with 85 mM sucrose (∘); 85 mM trehalose (□); 161 mMsucrose (□) or 161 mM trehalose (▴). Samples were lyophilized and storedat 30° C. followed by reconstitution with 0.9% benzyl alcohol to 100mg/mL antibody in 20 mM histidine at pH 6 with isotonic (340 mM) andhypertonic (644 mM) sugar concentration.

FIG. 19 illustrates aggregation of rhuMAb E25 formulated at 25 mg/mLinto 5 mM histidine at pH 6 with 85 mM sucrose (∘); 85 mM trehalose (□);161 mM sucrose (□) or 161 mM trehalose (▴). Samples were lyophilized andstored at 50° C. followed by reconstitution with 0.9% benzyl alcohol to100 mg/mL antibody in 20 mM histidine at pH 6 with isotonic (340 mM) andhypertonic (644 mM) sugar concentration.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

By “protein” is meant a sequence of amino acids for which the chainlength is sufficient to produce the higher levels of tertiary and/orquaternary structure. This is to distinguish from “peptides” or othersmall molecular weight drugs that do not have such structure. Typically,the protein herein will have a molecular weight of at least about 15-20kD, preferably at least about 20 kD.

Examples of proteins encompassed within the definition herein includemammalian proteins, such as, e.g., growth hormone, including humangrowth hormone and bovine growth hormone; growth hormone releasingfactor; parathyroid hormone; thyroid stimulating hormone; lipoproteins;a-l-antitrypsin; insulin A-chain; insulin B-chain; proinsulin; folliclestimulating hormone; calcitonin; luteinizing hormone; glucagon; clottingfactors such as factor VIIIC, factor IX, tissue factor, and vonWillebrands factor; anti-clotting factors such as Protein C; atrialnatriuretic factor; lung surfactant; a plasminogen activator, such asurokinase or tissue-type plasminogen activator (t-PA); bombazine;thrombin; tumor necrosis factor-α and -β; enkephalinase; RANTES(regulated on activation normally T-cell expressed and secreted); humanmacrophage inflammatory protein (MIP-1-α); serum albumin such as humanserum albumin; mullerian-inhibiting substance; relaxin A-chain; relaxinB-chain; prorelaxin; mouse gonadotropin-associated peptide; DNase;inhibin; activin; vascular endothelial growth factor (VEGF); receptorsfor hormones or growth factors; an integrin; protein A or D; rheumatoidfactors; a neurotrophic factor such as bone-derived neurotrophic factor(BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or anerve growth factor such as NGF-β; platelet-derived growth factor(PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growthfactor (EGF); transforming growth factor (TGF) such as TGF-α and TGF-β,including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growthfactor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I);insulin-like growth factor binding proteins; CD proteins such as CD3,CD4, CD8, CD19 and CD20; erythropoietin (EPO); thrombopoietin (TPO);osteoinductive factors; immunotoxins; a bone morphogenetic protein(BMP); an interferon such as interferon-α, -β, and -γ; colonystimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins(ILs), e.g., IL-1 to IL-10; superoxide dismutase; T-cell receptors;surface membrane proteins; decay accelerating factor (DAF); a viralantigen such as, for example, a portion of the AIDS envelope; transportproteins; homing receptors; addressins; regulatory proteins;immunoadhesins; antibodies; and biologically active fragments orvariants of any of the above-listed polypeptides.

The protein which is formulated is preferably essentially pure anddesirably essentially homogeneous (i.e. free from contaminating proteinsetc). “Essentially pure” protein means a composition comprising at leastabout 90% by weight of the protein, based on total weight of thecomposition, preferably at least about 95% by weight. “Essentiallyhomogeneous” protein means a composition comprising at least about 99%by weight of protein, based on total weight of the composition.

In certain embodiments, the protein is an antibody. The antibody maybind to any of the above-mentioned molecules, for example. Exemplarymolecular targets for antibodies encompassed by the present inventioninclude CD proteins such as CD3, CD4, CD8, CD19, CD20 and CD34; membersof the HER receptor family such as the EGF receptor, HER2, HER3 or HER4receptor; cell adhesion molecules such as LFA-1, Mol, p150,95, VLA-4,ICAM-1, VCAM and αv/β3 integrin including either α or β subunits thereof(e.g. anti-CD11a, anti-CD18 or anti-CD11b antibodies); growth factorssuch as VEGF; IgE; blood group antigens; flk2/flt3 receptor; obesity(OB) receptor; protein C etc.

The term “antibody” is used in the broadest sense and specificallycovers monoclonal antibodies (including full length antibodies whichhave an immunoglobulin Fc region), antibody compositions withpolyepitopic specificity, bispecific antibodies, diabodies, andsingle-chain molecules, as well as antibody fragments (e.g., Fab,F(ab′)₂, and Fv).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al., Nature, 256: 495 (1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991),for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementaritydetermining region (CDR) of the recipient are replaced by residues froma CDR of a non-human species (donor antibody) such as mouse, rat orrabbit having the desired specificity, affinity, and capacity. In someinstances, Fv framework region (FR) residues of the human immunoglobulinare replaced by corresponding non-human residues. Furthermore, humanizedantibodies may comprise residues which are found neither in therecipient antibody nor in the imported CDR or framework sequences. Thesemodifications are made to further refine and optimize antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin sequence. The humanizedantibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature,321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol., 2:593-596 (1992). The humanizedantibody includes a Primatized™ antibody wherein the antigen-bindingregion of the antibody is derived from an antibody produced byimmunizing macaque monkeys with the antigen of interest.

A “stable” formulation is one in which the protein therein essentiallyretains its physical and chemical stability and integrity upon storage.Various analytical techniques for measuring protein stability areavailable in the art and are reviewed in Peptide and Protein DrugDelivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y.,Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993).Stability can be measured at a selected temperature for a selected timeperiod. For rapid screening, the formulation may be kept at 40° C. for 2weeks to 1 month, at which time stability is measured. Where theformulation is to be stored at 2-8° C., generally the formulation shouldbe stable at 30° C. or 40° C. for at least 1 month and/or stable at 2-8°C. for at least 2 years. Where the formulation is to be stored at 30°C., generally the formulation should be stable for at least 2 years at30° C. and/or stable at 40° C. for at least 6 months. For example, theextent of aggregation following lyophilization and storage can be usedas an indicator of protein stability (see Examples herein). For example,a “stable” formulation may be one wherein less than about 10% andpreferably less than about 5% of the protein is present as an aggregatein the formulation. In other embodiments, any increase in aggregateformation following lyophilization and storage of the lyophilizedformulation can be determined. For example, a “stable” lyophilizedformulation may be one wherein the increase in aggregate in thelyophilized formulation is less than about 5% and preferably less thanabout 3%, when the lyophilized formulation is stored at 2-8° C. for atleast one year. In other embodiments, stability of the proteinformulation may be measured using a biological activity assay (see,e.g., Example 2 below).

A “reconstituted” formulation is one which has been prepared bydissolving a lyophilized protein formulation in a diluent such that theprotein is dispersed in the reconstituted formulation. The reconstitutedformulation in suitable for administration (e.g. parenteraladministration) to a patient to be treated with the protein of interestand, in certain embodiments of the invention, may be one which issuitable for subcutaneous administration.

By “isotonic” is meant that the formulation of interest has essentiallythe same osmotic pressure as human blood. Isotonic formulations willgenerally have an osmotic pressure from about 250 to 350 mOsm.Isotonicity can be measured using a vapor pressure or ice-freezing typeosmometer, for example.

A “lyoprotectant” is a molecule which, when combined with a protein ofinterest, significantly prevents or reduces chemical and/or physicalinstability of the protein upon lyophilization and subsequent storage.Exemplary lyoprotectants include sugars such as sucrose or trehalose; anamino acid such as monosodium glutamate or histidine; a methylamine suchas betaine; a lyotropic salt such as magnesium sulfate; a polyol such astrihydric or higher sugar alcohols, e.g. glycerin, erythritol, glycerol,arabitol, xylitol, sorbitol, and mannitol; propylene glycol;polyethylene glycol; Pluronics; and combinations thereof. The preferredlyoprotectant is a non-reducing sugar, such as trehalose or sucrose.

The lyoprotectant is added to the pre-lyophilized formulation in a“lyoprotecting amount” which means that, following lyophilization of theprotein in the presence of the lyoprotecting amount of thelyoprotectant, the protein essentially retains its physical and chemicalstability and integrity upon lyophilization and storage.

The “diluent” of interest herein is one which is pharmaceuticallyacceptable (safe and non-toxic for administration to a human) and isuseful for the preparation of a reconstituted formulation. Exemplarydiluents include sterile water, bacteriostatic water for injection(BWFI), a pH buffered solution (e.g. phosphate-buffered saline), sterilesaline solution, Ringer's solution or dextrose solution.

A “preservative” is a compound which can be added to the diluent toessentially reduce bacterial action in the reconstituted formulation,thus facilitating the production of a multi-use reconstitutedformulation, for example. Examples of potential preservatives includeoctadecyldimethylbenzyl ammonium chloride, hexamethonium chloride,benzalkonium chloride (a mixture of alkylbenzyldimethylammoniumchlorides in which the alkyl groups are long-chain compounds), andbenzethonium chloride. Other types of preservatives include aromaticalcohols such as phenol, butyl and benzyl alcohol, alkyl parabens suchas methyl or propyl paraben, catechol, resorcinol, cyclohexanol,3-pentanol, and m-cresol. The most preferred preservative herein isbenzyl alcohol.

A “bulking agent” is a compound which adds mass to the lyophilizedmixture and contributes to the physical structure of the lyophilizedcake (e.g. facilitates the production of an essentially uniformlyophilized cake which maintains an open pore structure). Exemplarybulking agents include mannitol, glycine, polyethylene glycol andxorbitol.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those in which the disorder is to beprevented.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal is human.

A “disorder” is any condition that would benefit from treatment with theprotein. This includes chronic and acute disorders or diseases includingthose pathological conditions which predispose the mammal to thedisorder in question. Non-limiting examples of disorders to be treatedherein include carcinomas and allergies.

II. Modes for Carrying out the Invention

A. Protein Preparation

The protein to be formulated is prepared using techniques which are wellestablished in the art including synthetic techniques (such asrecombinant techniques and peptide synthesis or a combination of thesetechniques) or may be isolated from an endogenous source of the protein.In certain embodiments of the invention, the protein of choice is anantibody. Techniques for the production of antibodies follow.

(i) Polyclonal Antibodies.

Polyclonal antibodies are generally raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining 1 mg or 1 μg of the peptide or conjugate (forrabbits or mice, respectively) with 3 volumes of Freund's completeadjuvant. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

(ii) Monoclonal Antibodies.

Monoclonal antibodies are obtained from a population of substantiallyhomogeneous antibodies, i.e., the individual antibodies comprising thepopulation are identical except for possible naturally occurringmutations that may be present in minor amounts. Thus, the modifier“monoclonal” indicates the character of the antibody as not being amixture of discrete antibodies.

For example, the monoclonal antibodies may be made using the hybridomamethod first described by Kohler et al., Nature, 256:495 (1975), or maybe made by recombinant DNA methods (U.S. Pat. No. 4,816,567). In thehybridoma method, a mouse or other appropriate host animal, such as ahamster, is immunized as hereinabove described to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the protein used for immunization. Alternatively,lymphocytes may be immunized in vitro. Lymphocytes then are fused withmyeloma cells using a suitable fusing agent, such as polyethyleneglycol, to form a hybridoma cell (Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2cells available from the American Type Culture Collection, Rockville,Md. USA. Human myeloma and mouse-human heteromyeloma cell lines alsohave been described for the production of human monoclonal antibodies(Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., MonoclonalAntibody Production Techniques and Applications, pp. 51-63 (MarcelDekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

The binding affinity of the monoclonal antibody can, for example, bedetermined by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies is readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of monoclonal antibodies in the recombinant hostcells. Review articles on recombinant expression in bacteria of DNAencoding the antibody include Skerra et al., Curr. Opinion in Immunol.,5:256-262 (1993) and Plückthun, Immunol. Revs., 130:151-188 (1992).

In a further embodiment, antibodies can be isolated from antibody phagelibraries generated using the techniques described in McCafferty et al.,Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991)and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe theisolation of murine and human antibodies, respectively, using phagelibraries. Subsequent publications describe the production of highaffinity (nM range) human antibodies by chain shuffling (Marks et al.,Bio/Technology, 10:779-783 (1992)), as well as combinatorial infectionand in vivo recombination as a strategy for constructing very largephage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266(1993)). Thus, these techniques are viable alternatives to traditionalmonoclonal antibody hybridoma techniques for isolation of monoclonalantibodies.

The DNA also may be modified, for example, by substituting the codingsequence for human heavy- and light-chain constant domains in place ofthe homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, etal., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by covalentlyjoining to the immunoglobulin coding sequence all or part of the codingsequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for theconstant domains of an antibody, or they are substituted for thevariable domains of one antigen-combining site of an antibody to createa chimeric bivalent antibody comprising one antigen-combining sitehaving specificity for an antigen and another antigen-combining sitehaving specificity for a different antigen.

Chimeric or hybrid antibodies also may be prepared in vitro using knownmethods in synthetic protein chemistry, including those involvingcrosslinking agents. For example, immunotoxins may be constructed usinga disulfide-exchange reaction or by forming a thioether bond. Examplesof suitable reagents for this purpose include iminothiolate andmethyl-4-mercaptobutyrimidate.

(iii) Humanized and Human Antibodies.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers(Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method uses a particular framework derived from theconsensus sequence of all human antibodies of a particular subgroup oflight or heavy chains. The same framework may be used for severaldifferent humanized antibodies (Carter et al., Proc. Natl. Acad. Sci.USA, 89:4285 (1992); Presta et al., J. Immnol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immuno., 7:33 (1993). Human antibodies can also be derivedfrom phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:381(1991); Marks et al., J. Mol. Biol., 222:581-597 (1991)).

(iv) Bispecific Antibodies

Bispecific antibodies (BsAbs) are antibodies that have bindingspecificities for at least two different epitopes. Such antibodies canbe derived from full length antibodies or antibody fragments (e.g.F(ab′)₂ bispecific antibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829 and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when the expression of at least two polypeptide chainsin equal ratios results in high yields or when the ratios are of noparticular significance.

In a preferred embodiment of this approach, the bispecific antibodiesare composed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690 published Mar. 3,1994. For further details of generating bispecific antibodies see, forexample, Suresh et al., Methods in Enzymology, 121:210 (1986).

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. The following techniques canalso be used for the production of bivalent antibody fragments which arenot necessarily bispecific. For example, Fab′ fragments recovered fromE. coli can be chemically coupled in vitro to form bivalent antibodies.See, Shalaby et al., J. Exp. Med., 175:217-225 (1992).

Various techniques for making and isolating bivalent antibody fragmentsdirectly from recombinant cell culture have also been described. Forexample, bivalent heterodimers have been produced using leucine zippers.Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucinezipper peptides from the Fos and Jun proteins were linked to the Fab′portions of two different antibodies by gene fusion. The antibodyhomodimers were reduced at the hinge region to form monomers and thenre-oxidized to form the antibody heterodimers. The “diabody” technologydescribed by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448(1993) has provided an alternative mechanism for makingbispecific/bivalent antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific/bivalentantibody fragments by the use of single-chain Fv (sFv) dimers has alsobeen reported. See Gruber et al., J. Immunol., 152:5368 (1994).

B. Preparation of the Lyophilized Formulation

After preparation of the protein of interest as described above, a“pre-lyophilized formulation” is produced. The amount of protein presentin the pre-lyophilized formulation is determined taking into account thedesired dose volumes, mode(s) of administration etc. Where the proteinof choice is an intact antibody (such as an anti-IgE or anti-HER2antibody), from about 2 mg/mL to about 50 mg/mL, preferably from about 5mg/mL to about 40 mg/mL and most preferably from about 20-30 mg/mL is anexemplary starting protein concentration. The protein is generallypresent in solution. For example, the protein may be present in apH-buffered solution at a pH from about 4-8, and preferably from about5-7. Exemplary buffers include histidine, phosphate, Tris, citrate,succinate and other organic acids. The buffer concentration can be fromabout 1 mM to about 20 mM, or from about 3 mM to about 15 mM, depending,for example, on the buffer and the desired isotonicity of theformulation (e.g. of the reconstituted formulation). The preferredbuffer is histidine in that, as demonstrated below, this can havelyoprotective properties. Succinate was shown to be another usefulbuffer.

The lyoprotectant is added to the pre-lyophilized formulation. Inpreferred embodiments, the lyoprotectant is a non-reducing sugar such assucrose or trehalose. The amount of lyoprotectant in the pre-lyophilizedformulation is generally such that, upon reconstitution, the resultingformulation will be isotonic. However, hypertonic reconstitutedformulations may also be suitable. In addition, the amount oflyoprotectant must not be too low such that an unacceptable amount ofdegradation/aggregation of the protein occurs upon lyophilization. Wherethe lyoprotectant is a sugar (such as sucrose or trehalose) and theprotein is an antibody, exemplary lyoprotectant concentrations in thepre-lyophilized formulation are from about 10 mM to about 400 mM, andpreferably from about 30 mM to about 300 mM, and most preferably fromabout 50 mM to about 100 mM.

The ratio of protein to lyoprotectant is selected for each protein andlyoprotectant combination. In the case of an antibody as the protein ofchoice and a sugar (e.g., sucrose or trehalose) as the lyoprotectant forgenerating an isotonic reconstituted formulation with a high proteinconcentration, the molar ratio of lyoprotectant to antibody may be fromabout 100 to about 1500 moles lyoprotectant to 1 mole antibody, andpreferably from about 200 to about 1000 moles of lyoprotectant to 1 moleantibody, for example from about 200 to about 600 moles of lyoprotectantto 1 mole antibody.

In preferred embodiments of the invention, it has been found to bedesirable to add a surfactant to the pre-lyophilized formulation.Alternatively, or in addition, the surfactant may be added to thelyophilized formulation and/or the reconstituted formulation. Exemplarysurfactants include nonionic surfactants such as polysorbates (e.g.polysorbates 20 or 80); poloxamers (e.g. poloxamer 188); Triton; sodiumdodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside;lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-,myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, orcetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g. Pluronics, PF68 etc). The amount ofsurfactant added is such that it reduces aggregation of thereconstituted protein and minimizes the formation of particulates afterreconstitution. For example, the surfactant may be present in thepre-lyophilized formulation in an amount from about 0.001-0.5%, andpreferably from about 0.005-0.05%.

In certain embodiments of the invention, a mixture of the lyoprotectant(such as sucrose or trehalose) and a bulking agent (e.g. mannitol orglycine) is used in the preparation of the pre-lyophilizationformulation. The bulking agent may allow for the production of a uniformlyophilized cake without excessive pockets therein etc.

Other pharmaceutically acceptable carriers, excipients or stabilizerssuch as those described in Remington's Pharmaceutical Sciences 16thedition, Osol, A. Ed. (1980) may be included in the pre-lyophilizedformulation (and/or the lyophilized formulation and/or the reconstitutedformulation) provided that they do not adversely affect the desiredcharacteristics of the formulation. Acceptable carriers, excipients orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed and include; additional buffering agents; preservatives;co-solvents; antioxidants including ascorbic acid and methionine;chelating agents such as EDTA; metal complexes (e.g. Zn-proteincomplexes); biodegradable polymers such as polyesters; and/orsalt-forming counterions such as sodium.

The formulation herein may also contain more than one protein asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect the otherprotein. For example, it may be desirable to provide two or moreantibodies which bind to the HER2 receptor or IgE in a singleformulation. Furthermore, anti-HER2 and anti-VEGF antibodies may becombined in the one formulation. Such proteins are suitably present incombination in amounts that are effective for the purpose intended.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes, prior to, or following, lyophilization and reconstitution.Alternatively, sterility of the entire mixture may be accomplished byautoclaving the ingredients, except for protein, at about 120° C. forabout 30 minutes, for example.

After the protein, lyoprotectant and other optional components are mixedtogether, the formulation is lyophilized. Many different freeze-dryersare available for this purpose such as Hull50™ (Hull, USA) or GT20™(Leybold-Heraeus, Germany) freeze-dryers. Freeze-drying is accomplishedby freezing the formulation and subsequently subliming ice from thefrozen content at a temperature suitable for primary drying. Under thiscondition, the product temperature is below the eutectic point or thecollapse temperature of the formulation. Typically, the shelftemperature for the primary drying will range from about −30 to 25° C.(provided the product remains frozen during primary drying) at asuitable pressure, ranging typically from about 50 to 250 mTorr. Theformulation, size and type of the container holding the sample (e.g.,glass vial) and the volume of liquid will mainly dictate the timerequired for drying, which can range from a few hours to several days(e.g. 40-60 hrs). A secondary drying stage may be carried out at about0-40° C., depending primarily on the type and size of container and thetype of protein employed. However, it was found herein that a secondarydrying step may not be necessary. For example, the shelf temperaturethroughout the entire water removal phase of lyophilization may be fromabout 15-30° C. (e.g., about 20° C.). The time and pressure required forsecondary drying will be that which produces a suitable lyophilizedcake, dependent, e.g., on the temperature and other parameters. Thesecondary drying time is dictated by the desired residual moisture levelin the product and typically takes at least about 5 hours (e.g. 10-15hours). The pressure may be the same as that employed during the primarydrying step. Freeze-drying conditions can be varied depending on theformulation and vial size.

In some instances, it may be desirable to lyophilize the proteinformulation in the container in which reconstitution of the protein isto be carried out in order to avoid a transfer step. The container inthis instance may, for example, be a 3, 5, 10, 20, 50 or 100cc vial.

As a general proposition, lyophilization will result in a lyophilizedformulation in which the moisture content thereof is less than about 5%,and preferably less than about 3%.

C. Reconstitution of the Lyophilized Formulation

At the desired stage, typically when it is time to administer theprotein to the patient, the lyophilized formulation may be reconstitutedwith a diluent such that the protein concentration in the reconstitutedformulation is at least 50 mg/mL, for example from about 50 mg/mL toabout 400 mg/mL, more preferably from about 80 mg/mL to about 300 mg/mL,and most preferably from about 90 mg/mL to about 150 mg/mL. Such highprotein concentrations in the reconstituted formulation are consideredto be particularly useful where subcutaneous delivery of thereconstituted formulation is intended. However, for other routes ofadministration, such as intravenous administration, lower concentrationsof the protein in the reconstituted formulation may be desired (forexample from about 5-50 mg/mL, or from about 10-40 mg/mL protein in thereconstituted formulation). In certain embodiments, the proteinconcentration in the reconstituted formulation is significantly higherthan that in the pre-lyophilized formulation. For example, the proteinconcentration in the reconstituted formulation may be about 2-40 times,preferably 3-10 times and most preferably 3-6 times (e.g. at least threefold or at least four fold) that of the pre-lyophilized formulation.

Reconstitution generally takes place at a temperature of about 25° C. toensure complete hydration, although other temperatures may be employedas desired. The time required for reconstitution will depend, e.g., onthe type of diluent, amount of excipient(s) and protein. Exemplarydiluents include sterile water, bacteriostatic water for injection(BWFI), a pH buffered solution (e.g. phosphate-buffered saline), sterilesaline solution, Ringer's solution or dextrose solution. The diluentoptionally contains a preservative. Exemplary preservatives have beendescribed above, with aromatic alcohols such as benzyl or phenol alcoholbeing the preferred preservatives. The amount of preservative employedis determined by assessing different preservative concentrations forcompatibility with the protein and preservative efficacy testing. Forexample, if the preservative is an aromatic alcohol (such as benzylalcohol), it can be present in an amount from about 0.1-2.0% andpreferably from about 0.5-1.5%, but most preferably about 1.0-1.2%.

Preferably, the reconstituted formulation has less than 6000 particlesper vial which are ≧10 μm in size.

D. Administration of the Reconstituted Formulation

The reconstituted formulation is administered to a mammal in need oftreatment with the protein, preferably a human, in accord with knownmethods, such as intravenous administration as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerobrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, topical, or inhalation routes.

In preferred embodiments, the reconstituted formulation is administeredto the mammal by subcutaneous (i.e. beneath the skin) administration.For such purposes, the formulation may be injected using a syringe.However, other devices for administration of the formulation areavailable such as injection devices (e.g. the Inject-ease™ and Genject™devices); injector pens (such as the GenPen™); needleless devices (e.g.MediJector™ and BioJector™); and subcutaneous patch delivery systems.

The appropriate dosage (“therapeutically effective amount”) of theprotein will depend, for example, on the condition to be treated, theseverity and course of the condition, whether the protein isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the protein, the type ofprotein used, and the discretion of the attending physician. The proteinis suitably administered to the patient at one time or over a series oftreatments and may be administered to the patient at any time fromdiagnosis onwards. The protein may be administered as the sole treatmentor in conjunction with other drugs or therapies useful in treating thecondition in question.

Where the protein of choice is an antibody, from about 0.1-20 mg/kg isan initial candidate dosage for administration to the patient, whether,for example, by one or more separate administrations. However, otherdosage regimens may be useful. The progress of this therapy is easilymonitored by conventional techniques.

In the case of an anti-HER2 antibody, a therapeutically effective amountof the antibody may be administered to treat or prevent cancercharacterized by overexpression of the HER2 receptor. It is contemplatedthat a reconstituted formulation of the anti-HER2 antibody may be usedto treat breast, ovarian, stomach, endometrial, salivary gland, lung,kidney, colon and/or bladder cancer. For example, the anti-HER2 antibodymay be used to treat ductal carcinoma in situ (DCIS). Exemplary dosagesof the anti-HER2 antibody are in the range 1-10 mg/kg by one or moreseparate administrations.

Uses for an anti-IgE formulation include the treatment or prophylaxis ofIgE-mediated allergic diseases, parasitic infections, interstitialcystitis and asthma, for example. Depending on the disease or disorderto be treated, a therapeutically effective amount (e.g. from about 1-15mg/kg) of the anti-IgE antibody is administered to the patient.

E. Articles of Manufacture

In another embodiment of the invention, an article of manufacture isprovided which contains the lyophilized formulation of the presentinvention and provides instructions for its reconstitution and/or use.The article of manufacture comprises a container. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Thecontainer holds the lyophilized formulation and the label on, orassociated with, the container may indicate directions forreconstitution and/or use. For example, the label may indicate that thelyophilized formulation is reconstituted to protein concentrations asdescribed above. The label may further indicate that the formulation isuseful or intended for subcutaneous administration. The containerholding the formulation may be a multi-use vial, which allows for repeatadministrations (e.g. from 2-6 administrations) of the reconstitutedformulation. The article of manufacture may further comprise a secondcontainer comprising a suitable diluent (e.g. BWFI). Upon mixing of thediluent and the lyophilized formulation, the final protein concentrationin the reconstituted formulation will generally be at least 50 mg/mL.The article of manufacture may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, syringes, and package inserts withinstructions for use.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All literature citations are incorporated byreference.

EXAMPLE 1 Anti-HER2 Formulation

Overexpression of the HER2 proto-oncogene product (p185^(HER2)) has beenassociated with a variety of aggressive human malignancies. The murinemonoclonal antibody known as muMAb4D5 is directed against theextracellular domain (ECD) of p185^(HER2). The muMAb4D5 molecule hasbeen humanized in an attempt to improve its clinical efficacy byreducing immunogenicity and allowing it to support human effectorfunctions (see WO 92/22653). This example describes the development of alyophilized formulation comprising full length humanized antibodyhuMAb4D5-8 described in WO 92/22653.

In the development of a lyophilized formulation, excipients and buffersare initially screened by measuring the stability of the protein afterlyophilization and reconstitution. The lyophilized protein in eachformulation is also subjected to accelerated stability studies todetermine the potential stability of the protein over its shelf-life.These accelerated studies are usually performed at temperatures abovethe proposed storage conditions and the data are then used to estimatethe activation energy for the degradation reactions assuming Arrheniuskinetics (Cleland et al., Critical Reviews in Therapeutic Drug CarrierSystems 10(4): 307-377 (1993)). The activation energy is then used tocalculate the expected shelf-life of the protein formulation at theproposed storage conditions.

In early screening studies, the stability of several lyophilizedrecombinant humanized anti-HER2 antibody (rhuMAb HER2) formulations wasinvestigated after incubation at 5° C. (proposed storage condition) and40° C. (accelerated stability condition). In the liquid state, rhuMAbHER2 was observed to degrade by deamidation (30Asn of light chain) andisoaspartate formation via a cyclic imide intermediate, succinimide(102Asp of heavy chain). The deamidation was minimized at pH 5.0resulting in degradation primarily at the succinimide. At pH 6.0,slightly greater deamidation was observed in the liquid proteinformulation. The lyophilized formulations were therefore studied with:(a) 5 or 10 mM succinate buffer, pH 5.0 or (b) 5 or 10 mM histidinebuffer, pH 6.0. Both buffers contained the surfactant, polysorbate 20(Tween 20™), which was employed to reduce the potential for aggregationof the reconstituted protein and minimize the formation of particulatesafter reconstitution. These buffers were used with and without varioussugars. The protein was formulated in the buffer at 5.0, 21.0 or 25.0mg/mL. These formulations were then lyophilized and assessed for proteinstability after 2 weeks at 5° C. and 40° C. In the lyophilizer, thevials were frozen at a shelf temperature of −55° C. for approximately 5hours followed by primary drying at a shelf temperature of 5° C. and 150mTorr for 30 hours, and drying to 1-2% residual moisture was achievedwith secondary drying at a shelf temperature of 20° C. for 10 hours. Themajor degradation route for this protein upon lyophilization wasaggregation, and therefore the protein stability was assessed by nativesize exclusion chromatography to measure the recovery of intact nativeprotein (% intact protein in Table 2 below).

The stabilizing effects of various lyoprotectant sugars on lyophilizedprotein was measured in 10 mM sodium succinate, pH 5.0 (Table 2). Athigh sugar concentrations (250-275 mM) and low protein concentration(5.0 mg/mL), trehalose and lactose stabilized the protein againstaggregation for the lyophilized protein stored for 2 weeks at 40° C.However, lactose, a reducing sugar, was observed to react with theprotein over longer term storage at 40° C. The formulations at 5.0 mg/mLprotein containing either sorbitol or mannitol yielded aggregatedprotein after storage at 40° C. for 2 weeks. At the higher proteinconcentration (21.0 mg/mL), formulations comprising mannitol, ormannitol in combination with sorbitol or glycine, contained aggregatedprotein after lyophilization and storage at both conditions. Incontrast, trehalose and sucrose prevented aggregation at both storageconditions.

The 250 mM trehalose and 250 mM lactose formulations were assessed forlong term stability. After 9 months at 40° C. or 12 months at 5° C.,there was no change in the % intact protein for the trehaloseformulation. For the lactose formulation, the % intact protein remainedconstant (same as initial) after 3 months at 40° C. or 6 months at 25°C. The trehalose formulation could be stored at controlled roomtemperature (15-30° C.) for 2 years without a significant change in %intact protein.

The 10 mM histidine, pH 6.0 formulation with mannitol contained lessaggregated protein after storage at 40° C. for 2 weeks than the 10 mMsuccinate formulation, pH 5.0 with mannitol. This result may be relatedto some stabilizing effect contributed by histidine alone. After storageat 40° C. for 2 weeks, there was, however, significant aggregation forhistidine alone or histidine/mannitol formulations. The addition ofsucrose at an equal mass to mannitol (10 mg/mL of each) in the histidineformulation stabilized the protein against aggregation for both storageconditions. The use of glycine with mannitol did not improve the proteinstability, while the sucrose/glycine formulation provided the samestability as the sucrose/mannitol formulation. These results furtherindicated that sucrose was useful for preventing aggregation of thelyophilized protein during storage.

TABLE 2 Composition Prior to Lyophilization % Intact Protein^(a)[Protein]^(b) Liquid Lyophilized Lyophilized (mg/mL) Formulation (5° C.)(2 wk, 5° C.) (2 wk, 40° C.) 10 mM sodium succinate pH 5.0 5.0 275 mMtrehalose, 0.01% Tween 98.9 99.1 98.9 20 ™ 5.0 275 mM lactose, 0.01%Tween 20 ™ 96.8 96.5 96.6 5.0 275 mM sorbitol, 0.01% Tween 99.4 99.395.4 20 ™ 5.0 250 mM mannitol, 0.01% Tween 100.0 99.9 98.8 20 ™ 5.0 250mM trehalose, 0.01% Tween 100.0 99.9 100.0 20 ™ 5.0 250 mM lactose,0.01% Tween 20 ™ 100.0 100.0 100.0 21.0 250 mM trehalose, 0.2% Tween99.3 99.1 99.1 20 ™ 21.0 250 mM sucrose, 0.2% Tween 20 ™ 99.6 99.6 99.721.0 250 mM mannitol, 0.01% Tween 100.0 94.6 94.0 20 ™ 21.0 188 mMmannitol/63 mM sorbitol, 99.8 98.6 96.5 0.01% Tween 20 ™ 21.0 250 mMmannitol/25 mM glycine, 99.5 96.5 96.4 0.01% Tween 20 ™ 10 mM histidinepH 6.0 21.0 No sugar, 0.01% Tween 20 ™ 100.0 99.9 98.9 21.0 54.9 mMmannitol, 0.01% Tween 100.0 99.9 99.2 20 ™ 21.0 29.2 mM sucrose/266.4 mMglycine, 100.0 100.0 99.6 0.01% Tween 20 ™ 21.0 54.9 mM mannitol/266.4mM 100.0 99.8 98.9 glycine, 0.01% Tween 20 ™ 21.0 54.9 mM mannitol/29.2mM 99.8 100.0 99.7 sucrose, 0.01% Tween 20 ™ ^(a)The fraction of intactprotein was measured by native size exclusion HPLC and the peak area ofthe native protein relative to the total peak area including aggregates(TSK3000 SW XL column, TosoHaas, with a flow rate of 1.0 mL/min; elutionwith phosphate buffered saline; detection at 214 and 280 nm). Theprotein formulations were analyzed before lyophilization (liquid, 5° C.)and after lyophilization and storage at 5° C. or 40° C. for 2 weeks.^(b)Formulations containing 5 mg/mL protein were reconstituted withdistilled water (20 mL, 5.0 mg/mL protein), and formulations containing21 mg/mL protein were reconstituted with bacteriostatic water forinjection (BWFI, 0.9% benzyl alcohol; 20 mL, 20 mg/mL protein).

The delivery of a high protein concentration is often required forsubcutaneous administration due to the volume limitations (≦1.5 mL) anddosing requirements (≧100 mg). However, high protein concentrations (≧50mg/mL) are often difficult to achieve in the manufacturing process sinceat high concentrations, the protein has a tendency to aggregate duringprocessing and becomes difficult to manipulate (e.g. pump) and sterilefilter. Alternatively, the lyophilization process may provide a methodto allow concentration of the protein. For example, the protein isfilled into vials at a volume (Vf) and then lyophilized. The lyophilizedprotein is then reconstituted with a smaller volume (Vr) of water orpreservative (e.g. BWFI) than the original volume (e.g. Vr=0.25Vf)resulting in a higher protein concentration in the reconstitutedsolution. This process also results in the concentration of the buffersand excipients. For subcutaneous administration, the solution isdesirably isotonic.

The amount of trehalose in the lyophilized rhuMAb HER2 was reduced toproduce an isotonic solution upon reconstitution to yield 100 mg/mLprotein. The stabilizing effect of trehalose was determined as afunction of concentration for 5 mM sodium succinate, pH 5.0 and 5 mMhistidine, pH 6.0 at 25.0 mg/mL protein (Table 3). At trehaloseconcentrations from 60 to 200 mM, there was no significant aggregationafter incubation of the lyophilized protein for 4 weeks at 40° C. Theseformulations were reconstituted with 20 mL of bacteriostatic water forinjection (BWFI, USP, 0.9% benzyl alcohol). Reconstitution of the 50 mMtrehalose formulation (5 mM sodium succinate) with 4 mL of BWFI (100mg/mL protein) after incubation for 4 weeks at 40° C. yielded a slightincrease in aggregate formation. The preserved reconstitutedformulations provided the advantage of multiple withdrawals from thesame vial without sterility concerns. When sterile needles are used,these formulations would then allow for several doses from a singlevial.

TABLE 3 Composition Prior to Lyophilization % Intact Protein^(a)[Protein] Liquid Lyophilized Lyophilized (mg/mL) Formulation (5° C.) (4wk, 5° C.) (4 wk, 40° C.) 5 mM sodium succinate pH 5.0 25.0 50 mMtrehalose, 0.01% Tween 20 ™^(b) 100.00 100.0 99.5 25.0 60 mM trehalose,0.01% Tween 20 ™ 100.0 100.0 99.9 25.0 60 mM trehalose, 0.01% Tween 20 ™100.0 100.0 99.2 25.0 100 mM trehalose, 0.01% Tween 100.0 100.0 99.720 ™ 25.0 150 mM trehalose, 0.01% Tween 100.0 100.0 99.8 20 ™ 25.0 200mM trehalose, 0.01% Tween 100.0 100.0 100.0 20 ™ 5 mM histidine pH 6.025.0 38.4 mM mannitol/20.4 mM sucrose, 100.0 100.0 99.3 0.01% Tween 20 ™25.0 38.4 mM mannitol/20.4 mM sucrose, 100.0 100.0 99.4 0.01% Tween20 ™^(c) 25.0 60 mM trehalose, 0.01% Tween 20 ™^(d) 100.0 100.0 99.825.0 60 mM trehalose, 0.01% Tween 20 ™ 100.0 100.0 99.4 25.0 100 mMtrehalose, 0.01% Tween 100.0 100.0 99.6 20 ™ 25.0 150 mM trehalose,0.01% Tween 100.0 100.0 100.0 20 ™ 25.0 200 mM trehalose, 0.01% Tween100.0 100.0 100.0 20 ™ ^(a)The fraction of intact protein was measuredby native size exclusion HPLC and defined as the peak area of the nativeprotein relative to the total peak area including aggregates (TSK3000 SWXL column, TosoHaas, with a flow rate of 1.0 mL/min; elution withphosphate buffered saline; detection at 214 and 280 nm). The proteinformulations were analyzed before lyophilization (liquid, 5° C.) andafter lyophilization and storage at 5° C. or 40° C. for 4 weeks.Formulations were reconstituted with bacteriostatic water for injection(BWFI, USP, 0.9% w/w benzyl alcohol; 20 mL, 22 mg/mL protein).^(b)Reconstituted with 4 mL of BWFI (0.9% benzyl alcohol) to yield 100mg/mL protein. ^(c)Reconstituted with 4 mL of BWFI (1.1% benzyl alcohol)to yield 100 mg/mL protein. ^(d)Sample incubated for 2 weeks at 5° C. or40° C. and then reconstituted with 20 mL of BWFI (0.9% benzyl alcohol)to yield 22 mg/mL protein.

Currently, rhuMAb HER2 is under investigation as a therapeutic for thetreatment of breast cancer. The protein is dosed to patients at 2 mg/kgon a weekly basis. Since the average weight of these patients is 65 kg,the average weekly dose is 130 mg of rhuMAb HER2. For subcutaneousadministration, injection volumes of 1.5 mL or less are well toleratedand, therefore, the protein concentration for a weekly subcutaneousadministration of rhuMAb HER2 may be approximately 100 mg/mL (130 mgaverage dose/1.5 mL). As mentioned above, this high proteinconcentration is difficult to manufacture and maintain in a stable form.To achieve this high protein concentration, rhuMAb HER2 formulated in:(a) 5 mM sodium succinate, pH 5.0 or (b) 5 mM histidine, pH 6.0, waslyophilized at 25 mg/mL protein in 60 mM trehalose, 0.01% Tween 20™. Thelyophilization was performed by filling 18 mL of the protein formulationinto 50 cc vials. In the lyophilizer, the vials were frozen at a shelftemperature of −55° C. for approximately 5 hours followed by primarydrying at a shelf temperature of 5° C. and 150 mTorr for 30 hours, anddrying to 1-2% residual moisture was achieved with secondary drying at ashelf temperature of 20° C. for 10 hours. Thermocouples placed in vialscontaining the placebo (formulation without protein) indicated that theproduct in the vials was maintained below −10° C. throughout primarydrying. Sequential stoppering studies during the lyophilization revealedthat the residual moisture after primary drying was usually less than10%.

The lyophilized protein was then reconstituted with either 4 or 20 mL ofBWFI (0.9 or 1.1% benzyl alcohol) to yield concentrated proteinsolutions:

-   -   (a) 4 mL: 102 mg/mL rhuMAb HER2, 245 mM trehalose, 21 mM sodium        succinate, pH 5.0 or 21 mM histidine, pH 6.0, 0.04% Tween 20™;    -   (b) 20 mL: 22 mg/mL rhuMAb HER2, 52 mM trehalose, 4 mM sodium        succinate, pH 5.0 or 4 mM histidine, pH 6.0, 0.009% Tween 20™.

After storage of the lyophilized formulations for 4 weeks at 40° C. andreconstitution to 22 mg/mL protein, the amount of aggregated proteinappeared to increase slightly with decreasing trehalose concentration.The stability of the lyophilized protein was not affected by the volumeof reconstitution. As shown in FIG. 1, the amount of intact proteinafter incubation of the lyophilized protein at 40° C. was the same forthe 60 mM trehalose, 5 mM sodium succinate, pH 5.0, 0.01% Tween 20™0formulation reconstituted with either 4 or 20 mL of BWFI.

The results shown in Table 3 suggested that there may be a relationshipbetween the trehalose concentration and the protein stability. Tofurther assess this relationship, the formulations containing differentconcentrations of trehalose formulated in either sodium succinate orhistidine were incubated for 91 days at 40° C. The stability was thenmeasured as a function of the trehalose to protein molar ratio for eachconcentration of trehalose. As shown in FIG. 2, the protein stabilityclearly decreased with decreasing trehalose concentration for bothformulations. There was no apparent difference between the two buffers,succinate and histidine, in these formulations suggesting that theprimary stabilizer under these conditions is trehalose. In addition, theobserved decrease in intact protein for both these formulations would beacceptable even at the low trehalose concentration for a formulationthat is stored at 2-8° C. throughout its shelf-life. However, ifcontrolled room temperature (30° C. maxmimum temperature) stability isrequired, higher trehalose concentrations (≧600:1 trehalose:protein) maybe needed depending on the stability specifications for the product(i.e. the specification for the amount of intact protein remaining after2 years of storage). Typically, a controlled room temperature storagecondition would require stability for 6 months at 40° C. which isequivalent to storage at 30° C. for 2 years.

As shown in FIG. 3, the 250 mM trehalose formulation was unchanged after6 months at 40° C. while both the 60 mM trehalose formulations were lessstable. The 60 mM trehalose formulations may then require refrigeratedstorage if the product specification at the end of its shelf-life is,for example, >98% intact protein by native size exclusionchromatography.

In the previous screening study, sucrose was also observed to preventaggregation of rhuMAb HER2 after lyophilization and subsequent storage.To achieve isotonic solutions after reconstitution for subcutaneousadministration (approximately four-fold concentration of formulationcomponents and protein), the sucrose concentration must be reducedsignificantly. The equal mass concentration of sucrose and mannitol(bulking agent) used in the screening studies prevented aggregation ofthe protein. A lower concentration of sucrose and mannitol (equal massconcentrations) was chosen as a potential subcutaneous formulation ofrhuMAb HER2. The protein solution (25 mg/mL protein, 5 mM histidine, pH6.0, 38.4 mM (7 mg/mL) mannitol, 20.4 mM (7 mg/mL) sucrose, 0.01% Tween20™) was lyophilized in the same manner as the 60 mM trehaloseformulation except that the primary drying cycle was extended to 54hours. After 4 weeks at 40° C., there was a slight increase in theamount of aggregates after reconstitution with 4.0 or 20.0 mL of BWFI(Table 3). The amount of aggregated protein was the same forreconstitution at 22 or 100 mg/mL protein (FIG. 4). Like the 60 mMtrehalose formulations, the mannitol/sucrose formulation yielded lessintact protein over time at 40° C. The molar ratio of sucrose to proteinfor this formulation was 120 to 1, indicating that the mannitol/sucrosecombination may be more effective than trehalose alone at the same molarratio of stabilizing sugar (FIGS. 2 and 4).

In the previous examples, the stability of the lyophilized rhuMAb HER2formulations was determined as a function of temperature. These studiesdemonstrated that the trehalose and mannitol/sucrose formulationsprevented degradation of the protein in the lyophilized state at hightemperatures (40° C.). However, these experiments did not address thestability of the protein after reconstitution and storage. Oncereconstituted with BWFI, the lyophilized rhuMAb HER2 formulations may beused for several administrations of the drug. In particular, the vialconfiguration (450 mg rhuMAb HER2) was designed to provide three dosesto the average patient (130 mg rhuMAb HER2 per dose). Since the drug isdosed weekly, the vial may be stored at least three weeks afterreconstitution. To assure that the rhuMAb HER2 remained stable afterreconstitution, stability studies on the reconstituted rhuMAb HER2formulations were performed at 5° C. and 25° C.

For subcutaneous administration, the formulations were reconstituted to100 mg/mL (4 mL BWFI). At this high protein concentration, the proteinmay be more susceptible to aggregation than the intravenous dosage formthat was reconstituted to 22 mg/mL protein (20 mL BWFI). The four rhuMAbHER2 formulations from the previous example were assessed foraggregation (loss of intact protein). As shown in Tables 4 through 6,there was no difference in stability for formulations reconstituted at22 and 100 mg/mL protein.

Furthermore, these formulations maintained the protein completely intactfor up to 90 days at 5° C. and 30 days at 25° C., indicating that thereconstituted protein could be stored refrigerated for at least 90 days.Unlike the lyophilized protein stability in the previous example, thetrehalose concentration in the formulation did not affect the proteinstability (Table 7).

TABLE 4 Stability of the reconstituted formulations for rhuMAb HER2lyophilized at 25 mg/mL protein in 5 mM sodium succinate, pH 5.0, 60 mMtrehalose, 0.01% Tween 20 ™ % Intact Protein 22 mg/mL 100 mg/mL Timeprotein protein (days) 5° C. 25° C. 5° C. 25° C. 0 99.9 99.9 99.7 99.714 ND 100.0 ND 100.0 30 100.0 100.0 100.0 100.0 91 99.8 ND 100 ND Thesamples were reconstituted with 4.0 or 20.0 mL of BWFI (1.1% or 0.9%benzyl alcohol), and then stored at 5° C. or 25° C. The % intact proteinwas defined as the fraction of native peak area as measured by nativesize exclusion chromatography. ND = not determined.

TABLE 5 Stability of the reconstituted formulations for rhuMAb HER2lyophilized at 25 mg/mL protein in 5 mM histidine, pH 6.0, 60 mMtrehalose, 0.01% Tween 20 ™ % Intact Protein 22 mg/mL 100 mg/mL Timeprotein protein (days) 5° C. 25° C. 5° C. 25° C. 0 100.0 100.0 100.0100.0 14 ND 100.0 ND 100.0 31 99.3 99.7 100.0 100.0 61 100.0 ND ND NDThe samples were reconstituted with 4.0 or 20.0 mL of BWFI (1.1% or 0.9%benzyl alcohol), and then stored at 5° C. or 25° C. The % intact proteinwas defined as the fraction of native peak area as measured by nativesize exclusion chromatography. ND = not determined.

TABLE 6 Stability of the reconstituted formulations for rhuMAb HER2lyophilized at 25 mg/mL protein in 5 mM histidine, pH 6.0, 38.4 mMmannitol, 20.4 mM sucrose, 0.01% Tween 20 ™ % Intact Protein 22 mg/mL100 mg/mL Time protein protein (days) 5° C. 25° C. 5° C. 25° C. 0 99.799.7 99.8 99.8 14 ND 100.0 ND 99.8 31 100.0 100.0 100.0 100.0 92 100.0ND 100.0 ND The samples were reconstituted with 4.0 or 20.0 mL of BWFI(1.1% or 0.9% benzyl alcohol), and then stored at 5° C. or 25° C. The %intact protein was defined as the fraction of native peak area asmeasured by native size exclusion chromatography. ND = not determined.

TABLE 7 Stability of the reconstituted formulations for rhuMAb HER2lyophilized at 21 mg/mL protein in 10 mM sodium succinate, pH 5.0, 250mM trehalose, 0.2% Tween 20 ™ % Intact Protein Time 21 mg/mL protein(days) 5° C. 25° C. 0 99.8 99.8 14 ND 100.0 31 99.9 99.4 92 99.8 ND Thesamples were reconstituted with 20.0 mL of BWFI (0.9% benzyl alcohol),and then stored at 5° C. or 25° C. The % intact protein was defined asthe fraction of native peak area as measured by native size exclusionchromatography. ND = not determined

As mentioned previously, the major degradation route for rhuMAb HER2 inaqueous solutions is deamidation or succinimide formation. The loss ofnative protein due to deamidation or succinimide formation was assessedfor the four reconstituted rhuMAb HER2 formulations.

Analysis of rhuMAb HER2 deamidation and succinimide formation wasperformed using cation exchange chromatography. A Bakerbond Wide-PoreCarboxy Sulfon (CSX) column (4.6×250 mm) was operated at a flow rate of1 mL/min. The mobile phase buffers were (A) 0.02 M sodium phosphate, pH6.9, and (B) 0.02 M sodium phosphate, pH 6.9, 0.2 M NaCl. Thechromatography was then performed at 40° C. as follows:

TABLE 8 Time (min) % Buffer B 0 10 55 45 57 100 62 100 62.1 10 63 10

Peak elution was monitored at 214 nm and 75 μg of protein was loaded foreach analysis.

Again, there were no differences in stability of the formulationsreconstituted at 22 and 100 mg/mL protein (FIGS. 5 through 7). Theprotein degradation was more rapid at 25° C. than 5° C. for eachformulation, and the rate of degradation was comparable for all theformulations stored at 5° C. The formulations containing histidineunderwent a slightly greater rate of degradation at 25° C. than thesuccinate formulations. The amount of trehalose in the formulation didnot affect the degradation rate at either temperature (FIGS. 5 and 8).These results indicated that these four formulations provide anacceptable rate of degradation under refrigerated storage conditions (5°C.) for the intended period of use (30 days after reconstitution withBWFI).

Multi-use formulations should pass preservative efficacy testing asdescribed by the US Pharmacopeia (USP) for use in the United States. TherhuMAb HER2 lyophilized formulation consisting of 25 mg/mL protein, 5 mMhistidine, pH 6.0, 60 mM trehalose, 0.01% Tween 20™0 was reconstitutedwith 20 mL of benzyl alcohol at concentrations between 0.9 and 1.5% w/w.For concentrations at or above 1.3% w/w, the reconstituted solutionbecame cloudy after overnight incubation at room temperature (˜25° C.).Reconstitution with the standard BWFI solution (0.9% benzyl alcohol)resulted in a solution that did not consistently pass the preservativechallenge tests. However, reconstitution with 1.0 or 1.1% benzyl alcoholwas both compatible with the formulation and passed the preservativechallenge testing. The manufacturer's specifications for the solutionrequired a range of ±10%, and therefore, the lyophilized formulationsare reconstituted with 1.1% benzyl alcohol (1.1±0.1%).

A single step lyophilization cycle for the rhuMAb HER2 formulation wasdeveloped. In the single step lyophilization cycle, rhuMAb HER2 at 25mg/mL, 60 mM trehalose, 5 mM histidine pH 6 and 0.01% polysorbate 20 waslyophilized at a shelf temperature of 20° C., and a pressure of 150mTorr. After 47 hours, the residual moisture content of the lyophilizedcake was less than 5%. This lyophilization cycle is considered to beuseful in that it simplifies the manufacturing process, by eliminatingthe secondary drying step.

EXAMPLE 2 Anti-IgE Formulation

IgE antibodies bind to specific high-affinity receptors on mast cells,leading to mast cell degranulation and release of mediators, such ashistamine, which produce symptoms associated with allergy. Hence,anti-IgE antibodies that block binding of IgE to its high-affinityreceptor are of potential therapeutic value in the treatment of allergy.These antibodies must also not bind to IgE once it is bound to thereceptor because this would trigger histamine release. This exampledescribes the development of a lyophilized formulation comprising fulllength humanized anti-IgE antibody MaE11 described in Presta et al. J.Immunology, 151: 2623-2632 (1993).

Materials: Highly purified rhuMAb E25 (recombinant humanized anti-IgEantibody MaE11) which did not contain Tween 20™ was used in theformulations described below. Spectra/Por 7 dialysis membranes werepurchased from Spectrum (Los Angeles, Calif.). All other reagents usedin this study were obtained from commercial sources and were ofanalytical grade. Formulation buffers and chromatography mobile phasewere prepared by mixing the appropriate amount of buffer and salt withMilli-Q water in a volumetric flask.

Formulation: E25 S Sepharose pool was dialyzed into formulation buffersas specified. Dialysis was accomplished by a minimum of 4×2L bufferexchanges over a 48 hour period at 2-8° C. Following dialysis,lyoprotectant was added at a isotonic concentration to some of theformulations as required. Protein concentration following dialysis wasdetermined by UV spectroscopy using a molar absorptivity of 1.60. Thedialyzed protein was diluted to the predetermined formulationconcentration with an appropriate formulation buffer, sterile filteredusing a 0.22 μm Millex-GV filter (Millipore) and dispensed intopre-washed and autoclaved glass vials. The vials were fitted withsiliconized teflon lyophilization stoppers and lyophilized using thefollowing conditions: the E25 formulation was frozen to −55° C. at 80°C./hour and the vial content was kept frozen for 4 hours. The shelftemperature was ramped to 25° C. at 10° C./hour for primary drying.Primary drying was carried out at 25° C., 50μ chamber vacuum pressurefor 39 hours such that the residual moisture of the lyophilized cake was1-2%. Following lyophilization, a vial of each formulation was removedfor t=0 analysis and the remaining vials were held at varioustemperatures which include −70° C., 2-8° C., 25° C., 30° C. (controlledroom temperature) 40° C. and 50° C.

Chromatography: Native size exclusion chromatography was carried out ona Bio-Rad Bio-Select™ SEC 250-5 column (300×7.8 mm) The column wasequilibrated and ran in PBS at a flow rate of 0.5 mL/min using a HewlettPackard 1090L HPLC equipped with a diode array detector. Molecularweight standards (Bio-Rad, Inc.) consisting of thyroglobulin (670 kd),gamma-globulin (158 kd), ovalbumin (44 kd), and cyanocobalamin (1.35 kd)were used to calibrate the column. The sample load was 25 μg and proteinwas detected by monitoring the UV absorption at 214 nm using Turbochrom3 software (PE Nelson, Inc).

Hydrophobic Interaction Chromatography: F(ab′)₂ fragments of the E25antibody were chromatographed using a TosoHaas Butyl-NPR column (3.5×4.6mm) and a Hewlett Packard 1090L HPLC equipped with a diode arraydetector. Elution buffer A was: 20 mM Tris, 2 M ammonium sulfate, 20%(v/v) glycerol, pH 8.0 while elution buffer B was: 20 mM Tris, 20% (v/v)glycerol, pH 8.0. The column was equilibrated with 10% elution buffer Bat a flow rate of 1.0 mL/min for a minimum of 20 minutes. The sampleload was 5 μg and protein was detected by monitoring the UV absorptionat 214 nm using Turbochrom 3 data acquisition software (PE Nelson, Inc).Following injection of the sample, the column was maintained at 10%buffer B for 1 minute followed by a linear gradient of from 10% to 62%buffer B in 20 minutes. The column was washed with 100% buffer B for 5minutes and re-equilibrated with 10% buffer B for a minimum of 20minutes between successive sample injections.

Antibody Binding Activity: IgE receptor binding inhibition assay(IE25:2) was carried out as described in Presta et al., supra, onsamples diluted to 20 μg/mL and 30 μg/mL in assay diluent (phosphatebuffered saline, 0.5% BSA, 0.05% polysorbate 20, 0.01% Thimerosol). Eachdilution was then assayed in triplicate and the results were multipliedby an appropriate dilution factor to yield an active concentration. Theresults from 6 assays were averaged. The assay measures the ability ofrhuMAb E25 to competitively bind to IgE and thereby prevent IgE frombinding to its high affinity receptor which is immobilized to an ELISAplate. The results are divided by the antibody concentration asdetermined by UV absorption spectroscopy and reported as a specificactivity. Previous experiments have shown that this assay is stabilityindicating.

Particulate Assay: Reconstituted vials of lyophilized rhuMAb E25 werepooled to achieve a volume of approximately 7 mL. A count of the numberof particles of size ranging from 2 to 80 μm present in 1 mL of samplewas determined using a Hiac/Royco model 8000 counter. The counter wasfirst washed with 1 mL of sample three times followed by the measurementof 1 mL of sample in triplicate. The instrument determines the number ofparticles per mL that are equal to or greater than 10 μm and the numberof particles per mL that are equal to or greater than 25 μm.

The first step in the development of a formulation for the anti-IgEantibody was to determine a suitable buffer and pH for lyophilizationand storage of the product. Antibody at a concentration of 5.0 mg/mL wasformulated into 10 mM succinate buffers ranging from pH 5.0 to pH 6.5and into sodium phosphate, potassium phosphate and histidine buffers atpH 7.0. FIG. 9 shows increased antibody aggregate was observed in thehigher pH formulations both before and after lyophilization. Anexception was the histidine formulation at pH 7, where no increase inaggregate was observed upon storage at 2-8° C. FIG. 10 shows rhuMAb E25lyophilized in 5 mM histidine buffer at both pH 6 and pH 7 and storedfor 1 year at 2-8° C., 25° C., and 40° C. At each assay time point andstorage temperature the pH 6 formulation had less aggregate than theantibody formulated at pH 7. These results show histidine at pH 6 is aparticularly useful buffer system for preventing aggregation of theantibody.

To facilitate screening of lyoprotectants, the anti-IgE antibody wasformulated into sodium succinate at pH 5 with or without alyoprotectant. Potential lyoprotectants, added at isotonicconcentrations, were grouped into 3 categories:

-   -   (a) non-reducing monosaccharide (i.e. mannitol);    -   (b) reducing disaccharides (i.e. lactose and maltose); and    -   (c) non-reducing disaccharides (i.e. trehalose and sucrose).

Aggregation of the formulations following storage at 2-8° C. and 40° C.for one year is shown in FIGS. 11 and 12. With storage at 2-8° C., themonosaccharide formulation (mannitol) aggregated at a rate similar tothe buffer control, while formulations containing the disaccharides wereequally effective in controlling aggregation (FIG. 11). The resultsfollowing storage at 40° C. where similar with the exception of thesucrose formulation which rapidly aggregated (which correlated with abrowning of the freeze-dried cake (FIG. 12)). This was later shown to becaused by degradation of sucrose following storage at both acidic pH andhigh temperature.

Hydrophobic interaction chromatography of the antibody formulated inhistidine buffer at pH 6 with lactose shows the antibody is alteredfollowing storage for 6 months at 40° C. (FIG. 13). The chromatographypeaks are broadened and the retention time decreases. These changes arenot observed with the buffer control and sucrose formulations storedunder similar conditions as shown in FIGS. 14 and 15, respectively.Furthermore, isoelectric focusing showed an acidic shift in the pI ofthe antibody formulated in lactose and stored at 25° C. and 40° C. Thisindicates that reducing sugars are not suitable as lyoprotectants forthe antibody.

Aggregation of lyophilized formulations of anti-IgE at a concentrationof 20 mg/mL in 5 mM histidine buffer at pH 6 with various concentrationsof sucrose and trehalose following storage for 12 weeks at 50° C. isshown in FIG. 16. Both sugars have a similar protective effect onaggregation when the sugar concentration is greater than 500 moles ofsugar per mole of antibody. From these results, isotonic and hypertonicformulations of both sucrose and trehalose were identified for furtherdevelopment. The formulations are designed to be filled prior tolyophilization at a relatively low concentration of antibody and thelyophilized product is reconstituted with less volume than was filledwith bacteriostatic water for injection (BWFI) comprising 0.9% benzylalcohol. This allows the concentration of the antibody immediately priorto subcutaneous delivery and includes a preservative for a potentialmulti-use formulation while avoiding interactions between the proteinand preservative upon long-term storage.

Isotonic formulation: Anti-IgE at 25 mg/mL formulated in 5 mM histidinebuffer at pH 6 with 500 moles of sugar per mole antibody which equals asugar concentration of 85 mM. This formulation is reconstituted withBWFI (0.9% benzyl alcohol) at a volume which is four times less than wasfilled. This results in a 100 mg/mL of antibody in 20 mM histidine at pH6 with an isotonic sugar concentration of 340 mM.

Hypertonic formulation: Anti-IgE at 25 mg/mL formulated in 5 mMhistidine buffer at pH 6 with 1000 moles of sugar per mole antibodywhich equals a sugar concentration of 161 mM. This formulation isreconstituted with BWFI (0.9% benzyl alcohol) at a volume which is fourtimes less than was filled. This results in a 100 mg/mL of antibody in20 mM histidine at pH 6 with a hypertonic sugar concentration of 644 mM.

Comparisons of the antibody aggregation following storage of theisotonic and hypertonic formulations for up to 36 weeks are shown inFIGS. 17 to 19. No change in aggregation is observed in either thehypertonic or isotonic formulations with storage at 2-8° C. (FIG. 17).With storage at controlled room temperature (30° C.) increasedaggregation is not observed in the hypertonic formulations while anincrease in aggregate of from 1 to 2% occurs in the isotonicformulations (FIG. 18). Finally, following storage at 50° C. a minimalincrease in aggregate is observed with the hypertonic formulations, a 4%increase in aggregate occurs with the isotonic trehalose formulation anda 12% increase in aggregate occurs with the isotonic sucrose formulation(FIG. 19). These results show the isotonic formulation contains theminimum amount of sugar necessary to maintain the stability of theantibody with storage at a temperature up to 30° C.

The binding activity of the anti-IgE in the isotonic and hypertonicformulations was measured in an IgE receptor inhibition assay. It wasdiscovered that the binding activity of the isotonic and hypertonicsucrose and trehalose formulations was essentially unchanged followingstorage at −70° C., 2-8° C., 30° C. and 50° C. for up to 36 weeks.

Lyophilized formulations of proteins are known to contain insolubleaggregates or particulates (Cleland et al., Critical Reviews inTherapeutic Drug Carrier Systems, 10 (4):307-377 (1993)). Accordingly, aparticulate assay of antibody lyophilized at a concentration of 25 mg/mLin 5 mM histidine, pH 6 with the addition of 85 mM and 161 mM sucroseand trehalose was performed. Polysorbate 20 was added to theformulations at a concentration of 0.005%, 0.01%, and 0.02%. Sampleswere lyophilized and assayed following reconstitution to 100 mg/mLantibody in 20 mM histidine, pH 6 with 340 mM and 644 mM sugar. Thepolysorbate 20 concentration following reconstitution was 0.02%, 0.04%,and 0.08%.

Table 9 below shows the number of particles of size equal to or greaterthan 10 μm and equal to or greater than 25 μm from the isotonic andhypertonic sucrose and trehalose formulations. Polysorbate 20 was addedto the formulations at concentrations of 0.005%, 0.01%, and 0.02% priorto lyophilization. The results show that the addition of Tween™ to theformulation significantly reduces the number of particles in each sizerange tested. The US Pharmacopeia (USP) specification for small volumeinjections are not more than 6,000 particles of greater than or equal to10 μm and not more than 600 particles of greater than or equal to 25 μmper container (Cleland et al., supra). With the addition of polysorbate20, both the hypertonic and isotonic formulations pass thisspecification.

TABLE 9 Particles per mL Formulation Polysorbate 20 ≧10 μm ≧25 μMIsotonic Sucrose None 16,122 28 0.005%  173 2 0.01% 224 5 0.02% 303 6Hypertonic Sucrose None 14,220 84 0.005%  73 6 0.01% 51 0 0.02% 6Isotonic Trehalose None 33,407 24 0.005%  569 4 0.01% 991 16 0.02% 605 9Hypertonic None 24,967 28 Trehalose 0.005%  310 11 0.01% 209 6 0.02% 3446

One formulation developed for the anti-IgE antibody (i.e. 143 mg vialisotonic formulation of rhuMAb E25) which is considered to be useful forsubcutaneous delivery of this antibody is shown in Table 10 below. A 10cc vial is filled with 5.7 mL of rhuMAb E25 at a concentration of 25mg/mL formulated in 5 mM histidine at pH 6.0 with 0.01% polysorbate 20.Sucrose is added as a lyoprotectant at a concentration of 85 mM whichcorresponds to a molar ratio of sugar to antibody of 500 to 1. The vialis lyophilized and reconstituted with 0.9% benzyl alcohol to one quarterof the volume of the fill or 1.2 mL. The final concentration ofcomponents in the formulation is increased four fold to 100 mg/mL rhuMAbE25 in 20 mM histidine at pH 6 with 0.04% polysorbate 20 and 340 mMsucrose (isotonic) and 0.9% benzyl alcohol. The formulation containshistidine buffer at pH 6 because of its demonstrated protective effecton antibody aggregation. Sucrose was added as the lyoprotectant becauseof previous use in the pharmaceutical industry. The concentration ofsugar was chosen to result in an isotonic formulation uponreconstitution. Finally, polysorbate 20 is added to prevent theformation of insoluble aggregates.

TABLE 10 Pre-lyophilized Formulation Reconstituted Formulation (Fill 5.7mL into 10 cc vial) (1.2 mL 0.9% Benzyl Alcohol) 25 mg/mL rhuMAb E25 100mg/mL rhuMAb E25  5 mM Histidine, pH 6.0  20 mM Histidine, pH 6.0 85 mMSucrose 340 mM Sucrose 0.01% Polysorbate 20 0.04% Polysorbate 20 — 0.9%Benzyl Alcohol

What is claimed is:
 1. A stable lyophilized pharmaceutical formulationcomprising a lyophilized mixture of a lyoprotectant and a monoclonalanti-HER2 antibody, wherein the lyoprotectant is trehalose or sucroseand the molar ratio of lyoprotectant: antibody is 200-600 molelyoprotectant: 1 mole antibody.
 2. The formulation of claim 1, whereinthe monoclonal anti-HER2 antibody is huMab4D5-8.
 3. The formulation ofclaim 1 or 2 comprising the anti-HER2 antibody in a pre-lyophilizedamount of 20-30 mg/mL, sucrose or trehalose in a pre-lyophilized amountof 40-80 mM, a buffer and a surfactant, wherein the buffer is histidineor succinate buffer.
 4. The formulation of claim 1 or 2, wherein thelyoprotectant is trehalose.
 5. The formulation of claim 3, wherein thelyoprotectant is trehalose.
 6. The formulation of claim 4, wherein themolar ratio of trehalose:antibody is 360:1.
 7. The formulation of claim5, wherein the molar ratio of trehalose:antibody is 360:1.
 8. Theformulation of claim 5, wherein the surfactant is polysorbate
 20. 9. Theformulation of claim 7, wherein the surfactant is polysorbate
 20. 10.The formulation of claim 8, which is lyophilized and stable at 40° C.for at least 1 month.
 11. The formulation of claim 9, which islyophilized and stable at 40° C. for at least 1 month.
 12. Theformulation of claim 3, which is reconstituted with a diluent such thatthe anti-HER2 antibody concentration in the reconstituted formulation is10-30mg/mL, wherein the reconstituted formulation is stable at 2-8° C.for at least 30 days.
 13. The formulation of claim 5, which isreconstituted with a diluent such that the anti-HER2 antibodyconcentration in the reconstituted formulation is 10-30 mg/mL, whereinthe reconstituted formulation is stable at 2-8° C. for at least 30days.14. The formulation of claim 9, which is reconstituted with a diluentsuch that the anti-HER2 antibody concentration in the reconstitutedformulation is 10-30 mg/mL, wherein the reconstituted formulation isstable at 2-8° C. for at least 30days.
 15. The formulation of claim 13,wherein the diluent is sterile water or bacteriostatic water forinjection (BWFI).
 16. The formulation of claim 14, wherein the diluentis sterile water or bacteriostatic water for injection (BWFI).